Abstract
Modified oligonucleotides continue to play an important role as antisense compounds that inhibit the expression of genes associated with metabolic disorders, cancer, and infectious diseases. Because the majority of modifications render these molecules refractory to standard enzymatic sequencing techniques, alternative sequencing methods which are fast and reliable are needed. In this work we explore how sugar and backbone modifications affect fragmentation patterns observed from oligonucleotides which are fragmented by infrared multiple photon dissociation in the external reservoir of an electrospray ionization Fourier transform ion cyclotron mass spectrometer. The modifications influence which fragment types (i.e., an-B versus cn) dominate and the ease with which the oligonucleotides are fragmented. General observations for confirming the sequence of oligonucleotides are described.
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Cook, P. D. Medicinal Chemistry Strategies for Antisense Research. Antisense Research and Applications; In Crooke, S. T.; Lebleu, B., Eds.; CRC Press: Boca Raton, 1993; 149–187.
Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray Ionization for Mass Spectrometry of Large Biomolecules. Science 1989, 246, 64–71.
Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. New Developments in Biochemical Mass Spectrometry: Electrospray Ionization. Anal. Chem. 1990, 62(9), 882–899.
Smith, R. D.; Loo, J. A.; Ogorzalek-Loo, R. R.; Busman, M.; Udseth, H. R. Principles and Practice of Electrospray Ionization Mass-Spectrometry for Large Polypeptides and Proteins. Mass Spectrom. Rev. 1991, 10(5), 359–451.
Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Matrix-Assisted Ultraviolet Laser Desorption of Non-Volatile Compounds. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53–68.
Bahr, U.; Karas, M.; Hillenkamp, F. Analysis of Biopolymers by Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. Fresenius J. Anal. Chem. 1994, 348(12), 783–791.
Little, D. P.; Aaserud, D. J.; Valaskovic, G. A.; McLafferty, F. W. Sequence Information from 42–108-mer DNAs (Complete for a 50-mer) by Tandem Mass Spectrometry. J. Am. Chem. Soc. 1996, 118(39), 9352–9359.
Little, D. P.; McLafferty, F. W. Sequencing 50-mer DNAs Using Electrospray Tandem Mass Spectrometry and Complementary Fragmentation Methods. J. Am. Chem. Soc. 1995, 117, 6783–6784.
Little, D. P.; Thannhauser, T. W.; McLafferty, F. W. Verification of 50- to 100-mer DNA and RNA Sequences with High-Resolution Mass Spectrometry. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2318–2322.
McLuckey, S. A.; Habibigoudarzi, S. Ion Trap-Tandem Mass Spectrometry Applied to Small Multiply Charged Oligonucleotides with a Modified Base. J. Am. Soc. Mass Spectrom. 1994, 5(8), 740–747.
Ni, J. Pomerantz, S. C. McCloskey, J. A. Rapid Sequencing of Modified Oligonucleotides Using Tandem Mass Sectrometry with Electrospray Ionization. Nucleic Acids Symposium Series. Proceedings of the 23rd Symposium on Nucleic Acids Chemistry; 1996; pp 113–114.
Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McCloskey, J. A. Interpretation of Oligonucleotide Mass Spectra for Determination of Sequence Using Electrospray Ionization and Tandem Mass Spectrometry. Anal. Chem. 1996, 68(13), 1989–1999.
Barry, J. P.; Vouros, P.; Vanschepdael, A.; Law, S. J. Mass and Sequence Verification of Modified Oligonucleotides Using Electrospray Tandem Mass Spectrometry. J. Mass Spectrom. 1995, 30(7), 993–1006.
Hofstadler, S. A.; Sannes-Lowery, K. A.; Griffey, R. H. Infrared Multiphoton Dissociation in an External Ion Reservoir. Anal. Chem. 1999, 71, 2067–2070.
Anderson, G. A.; Bruce, J. E. ICR 2LS version 2.18. Pacific Northwest National Laboratory: Richland, WA, 1996.
McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. Tandem Mass Spectrometry of Small, Multiply Charged Oligonucleotides. J. Am. Soc. Mass Spectrom. 1992, 3(1), 60–70.
Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. Rapid Sequencing of Oligonucleotides by High-Resolution Mass Spectrometry. J. Am. Chem. Soc. 1994, 116(11), 4893–4897.
Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Infrared Multiphoton Dissociation of Large Multiply Charged Ions for Biomolecule Sequencing. Anal. Chem. 1994, 66(18), 2809–2815.
Aaserud, D. J.; Guan, Z.; Little, D. P.; McLafferty, F. W. DNA Sequencing with Blackbody Infrared Radiative Dissociation of Electrosprayed Ions. Int. J. Mass Spectrom. Ion Processes 1997, 167(168), 705–712.
Sannes-Lowery, K. A.; Hofstadler, S. A. Characterization of Multipole Storage Assisted Dissociation: Implications for Electrospray Ionization Mass Spectrometry Characterization of Biomolecules. J. Am. Soc. Mass Spectrom. 2000, 11(1), 1–9.
Tang, W.; Zhu, L.; Smith, L. M. Controlling DNA Fragmentation in MALDI-MS by Chemical Modification. Anal. Chem. 1997, 69(3), 302–312.
Wang, Y.; Taylor, J.-S.; Gross, M. L. Differentiation of Isomeric Photomodified Oligodeoxynucleotides by Fragmentation of Ions Produced by Matrix-Assisted Laser Desorption Ionization and Electrospray Ionization. J. Am. Soc. Mass Spectrom. 1999, 10(4), 329–338.
Hannis, J. C.; Muddiman, D. C. Tailoring the gas-phase dissociation and determining the relative energy of activation for dissociation of 7-deaza purine modified oligonucleotides containing a repeating motif. Int. J. of Mass Spectrom. 2002, 219(1), 139–150.
Wang, Y.; Men, L.; Vivekananda, S. Fragmentation of Deprotonated Ions of Oligodeoxynucleotides Carrying a 5-Formyluracil or 2-Aminoimidazolone. J. Am. Soc. Mass Spectrom. 2002, 13, 1190–1194.
Bartlett, M. G.; McCloskey, J. A.; Manalili, S.; Griffey, R. H. The Effect of Backbone Charge on the Collision-Induced Dissociation of Oligonucleotides. J. Mass Spectrom. 1996, 31(11), 1277–1283.
Wan, K. X.; Gross, M. L. Fragmentation Mechanisms of Oligodeoxynucleotides: Effects of Replacing Phosphates with Methylphosphonates and Thymines with Other Bases in T-Rich Sequences. J. Am. Soc. Mass Spectrom. 2001, 12(5), 580–589.
Nordhoff, E.; Karas, M.; Cramer, R.; Hahner, S.; Hillenkamp, F.; Kirpekar, F.; Lezius, A.; Muth, J.; Meier, C.; Engels, J. W. Direct Mass Spectrometric Sequencing of Low-Picomole Amounts of Oligodeoxynucleotides with up to 21 Bases by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. J. Mass Spectrom. 1995, 30(1), 99–112.
Wang, Z.; Wan, K. X.; Ramanathan, R.; Taylor, J. S.; Gross, M. L. Structure and Fragmentation Mechanisms of Isomeric T-Rich Oligodeoxynucleotides: A Comparison of Four Tandem Mass Spectrometric Methods. J. Am. Soc. Mass Spectrom. 1998, 9(7), 683–691.
Wan, K. X.; Gross, J.; Hillenkamp, F.; Gross, M. L. Fragmentation Mechanisms of Oligodeoxynucleotides Studied by H/D Exchange and Electrospray Ionization Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12(2), 193–205.
Gross, J.; Hillenkamp, F.; Wan, K. X.; Gross, M. L. Metastable Decay of Negatively Charged Oligodeoxynucleotides Analyzed with Ultraviolet Matrix-Assisted Laser Desorption/Ionization Post-Source Decay and Deuterium Exchange. J. Am. Soc. Mass Spectrom. 2001, 12(2), 180–192.
Altmann, K.-H.; Dean, N. M.; Fabbro, D.; Freier, S. M.; Geiger, T.; Haener, R.; Huesken, D.; Martin, P.; Monia, B. P. Second Generation of Antisense Oligonucleotides. From Nuclease Resistance to Biological Efficacy in Animals. Chimia 1996, 4, 168–176.
McLuckey, S. A.; Habibi-Goudarzi, S. Decompositions of Multiply Charged Oligonucleotide Anions. J. Am. Chem. Soc. 1993, 115, 12085–12095.
Rodgers, M. T.; Campbell, S.; Marzluff, E. M.; Beauchamp, J. L. Low-Energy Collision-Induced Dissociation of Deprotonated Dinucleotides: Determination of the Energetically Favored Dissociation Pathways and the Relative Acidities of the Nucleic Acid Bases. Int. J. Mass Spectrom. Ion Processes 1994, 137, 121–149.
Garrett, E. R.; Seydel, J. K.; Sharpen, A. J. The Acid-Catalyzed Solvolysis of Pyrimidine Nucleosides. J. Org. Chem. 1966, 31(7), 2219–2227.
Garrett, E. R.; Mehta, P. J. Solvolysis of Adenine Nucleosides. II. Effects of Sugars and Adenine Substituents on Alkaline Solvolyses. J. Am. Chem. Soc. 1972, 94(24), 8542–8547.
Garrett, E. R.; Mehta, P. J. Solvolysis of Adenine Nucleosides. I. Effects of Sugars and Adenine Substituents on Acid Solvolyses. J. Am. Chem. Soc. 1972, 94(24), 8532–8541.
York, J. L. Effect of the Structure of the Glycon on the Acid-Catalyzed Hydrolysis of Adenine Nucleosides. J. Org. Chem. 1981, 46(10), 2171–2173.
Schurch, S.; Bernal-Mendez, E.; Leumann, C. J. Electrospray Tandem Mass Spectrometry of Mixed-Sequence RNA/DNA Oligonucleotides. J. Am. Soc. Mass Spectrom. 2002, 13(8), 936–945.
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Published online June 23, 2003
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Sannes-Lowery, K.A., Hofstadler, S.A. Sequence confirmation of modified oligonucleotides using IRMPD in the external ion reservoir of an electrospray ionization fourier transform ion cyclotron mass spectrometer. J Am Soc Mass Spectrom 14, 825–833 (2003). https://doi.org/10.1016/S1044-0305(03)00335-0
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DOI: https://doi.org/10.1016/S1044-0305(03)00335-0